PROJECT SUMMARY Cardiovascular Disease (CVD) is the number one cause of death for both men and women in the US. However, the prevalence of CVD in women at advanced ages actually outnumbers men, and women generally face a worse prognosis following a primary CVD event. Thus, there is a critical need for identifying and understanding the sex-specific biological mechanisms that underlie the development of CVD in order to reduce the morbidity and mortality associated with CVD, particularly in women. In this regard, our recent metabolomics and genetic analyses in a cohort of ~10,000 CVD patients, followed by independent replication in >53,000 subjects, led to the discovery that one of the major genetic determinants of plasma glycine levels is strongly associated with 12% reduced risk of CVD in women (p=6.3x10-5) but not men (p=0.95). The lead variant underlying this striking sex-specific association is located in carbamoyl phosphate synthase 1 (CPS1), which encodes the rate-limiting enzyme in the urea cycle, and the athero-protective allele is associated with increased glycine levels and decreased urea cycle metabolites. This novel finding represents one of the first sexually dimorphic associations reported in the literature for CVD in either men or women, and the magnitude of its effect is equivalent to the most strongly associated loci identified thus far for CVD. However, epidemiological or experimental data directly linking glycine levels with CVD are lacking, and additional studies are needed to prove that glycine metabolism is causally and inversely related to the development of atherosclerosis. The overall goals of our application are to address these fundamentally important gaps in knowledge. We hypothesize that glycine metabolism represents a novel sex-specific, protective, and causal pathway for CVD. To investigate this hypothesis, we propose integrative clinical, genetics, and bioinformatics approaches in human populations, complemented with studies that leverage targeted genetic perturbation and dietary manipulation in animal models. In Specific Aim 1, we will determine the clinical association of glycine- related metabolites with prevalent and incident CVD phenotypes in two independent human cohorts (n>6000), and test whether these associations are modulated by female sex hormones. In parallel, we will conduct the largest meta-analyses of genome-wide association study (GWAS) data to date for these metabolites (n>12,600) and determine whether newly identified loci exhibit sex-specific associations with risk of CVD by leveraging GWAS results from the CARDIoGRAM Consortium (n~185,000). In Specific Aim 2, we will characterize the sex-specific metabolomics profile, atherosclerosis susceptibility, and functional/mechanistic consequences of dietary glycine supplementation or Cps1 deficiency in mice. Taken together, the proposed studies will leverage novel biomarker measures in independent human cohorts, already existing large-scale GWAS data, newly developed mouse models, and ex vivo functional studies to reveal the sexually dimorphic genetic architecture of glycine metabolism and determine its biologically causal relationship with CVD.